Inhibition of neointimal hyperplasia in a rabbit vein graft model following non-viral transfection with human iNOS cDNA.

Vein graft failure caused by neointimal hyperplasia (IH) after coronary artery bypass grafting with saphenous veins is a major clinical problem. The lack of safe and efficient vectors for vascular gene transfer has significantly hindered progress in this field. We have developed a Receptor-Targeted Nanocomplex (RTN) vector system for this purpose and assessed its therapeutic efficacy in a rabbit vein graft model of bypass grafting. Adventitial delivery of ß-Galactosidase showed widespread transfection throughout the vein wall on day 7, estimated at about 10% of cells in the adventitia and media. Vein grafts were then transfected with a plasmid encoding inducible nitric oxide synthase (iNOS) and engrafted into the carotid artery. Fluorescent immunohistochemistry analysis of samples from rabbits killed at 7 days after surgery showed that mostly endothelial cells and macrophages were transfected. Morphometric analysis of vein graft samples from the 28-day groups showed approximately a 50% reduction of neointimal thickness and 64% reduction of neointimal area in the iNOS-treated group compared with the surgery control groups. This study demonstrates efficacy of iNOS gene delivery by the RTN formulation in reducing IH in the rabbit model of vein graft disease.

The effect of transforming growth factor beta on rates of procollagen synthesis and degradation in vitro.

Transforming growth factor beta (TGF beta) is known to stimulate procollagen production and steady-state levels of procollagen mRNAs, but its ability to affect post-translational processing of procollagen has been little studied. This paper demonstrates the application of recently developed ultrasensitive methods for measuring hydroxyproline to assess rates of procollagen synthesis and degradation in vitro with and without TGF beta. Foetal rat fibroblasts synthesized 8.63 +/- 0.21 pmol hydroxyproline/micrograms DNA per h, which corresponds to approx. 40 molecules of procollagen/cell per s. Addition of TGF beta to cultures increased total amounts of procollagen synthesized and degraded by 112% and 82%, respectively, but there was a significant decrease in the proportion of procollagen degraded (control, 38.0 +/- 1.1%; TGF beta, 32.3 +/- 0.9%; P less than 0.005). This study demonstrates a novel mechanism which may contribute to the TGF beta-induced increase in procollagen production by fibroblasts.

Cytokine regulation of mesothelial cell proliferation in vitro and in vivo.

Previous studies have demonstrated mitogenic effects of several mediators on mesothelial cells in vitro, but their effects in vivo have not been investigated. The aim of this study was to examine the effects of various cytokines on normal mesothelial cell proliferation in vitro and in vivo and correlate the findings in both assay systems. In vitro proliferation was assessed using a technique based on the uptake and subsequent release of methylene blue. Autoradiographic methods were applied in a murine model to assess mitogenic activity of these factors on mesothelium in vivo. In vitro data demonstrated a dose-dependent increase in human mesothelial cell proliferation by all mediators examined: at optimal concentrations, proliferation was enhanced between 26.53 +/- 3.77% standard deviation (SD), p < 0.001 for fibroblast growth factor-2 (FGF-2) and 114.58 +/- 6.97%, p < 0.001 for platelet-derived growth factor-AB (PDGF-AB) above control medium. In vivo, DNA synthesis in mesothelial cells was stimulated by FGF-2 (29.52 +/- 5.85% labeled cells, compared with 7.04 +/- 4.36% for control medium; p < 0.001), tumor necrosis factor-alpha (TNF-alpha; 13.14 +/- 4.55% compared with 7.23 +/- 2.85; p < 0.005) and PDGF-BB (11.53 +/- 4.74% compared with 4.67 +/- 3.48%; p < 0.005). Transforming growth factor-beta1 (TGF-beta1) and epidermal growth factor (EGF) had no effect on DNA synthesis in mesothelial cells in vivo. It is concluded that FGF2, TNF-alpha, and PDGF stimulate mesothelial cell proliferation in vitro and in vivo, whereas TGF-beta1 and EGF only had a mitogenic effect in vitro at the concentrations examined. The mitogenic potency of the different PDGF isoforms in vitro was consistent with PDGF-alpha and beta receptor expression.

The pathogenesis of pulmonary fibrosis: is there a fibrosis gene?

Interstitial fibrosis is seen in the lung in response to a variety of insults, and often appears stereotypical in terms of its clinical and pathological features. However, exposure to a known aetiological factor does not always lead to fibrosis. For example in bleomycin-induced pulmonary fibrosis, a wide variation in response is seen both in humans and in animal models, which is not completely accounted for by known risk factors. These observations and the existence of a number of familial forms of lung fibrosis suggest a genetic predisposition. Current hypotheses concerning the pathogenesis of pulmonary fibrosis propose an initial stage involving the influx of inflammatory cells into the interstitium. These cells, together with activated resident cells are then thought to release polypeptide mediators that stimulate the fibroblast proliferation and matrix protein synthesis typical of these disorders. Genetic influences could have an important role in regulating a number of these events, altering the immunological response to injury or modulating collagen metabolism in the lung. However, despite recent advances in molecular genetic techniques, there have been few human studies to date. Most have concentrated on genetic loci with a high degree of polymorphism such as the human leucocyte antigen (HLA) system and yield conflicting results. Others offer tantalising but as yet, incomplete insights into the mechanisms involved. Defining the genetic abnormalities underlying both the familial forms of pulmonary fibrosis and the variations seen in response to lung injury should enhance our understanding of the pathogenic processes and help to focus research in this area.

Age-related alterations in collagen and total protein metabolism determined in cultured rat dermal fibroblasts: age-related trends parallel those observed in rat skin in vivo.

The cultured fibroblast has been extensively used as a model system to study aging. However, few studies have examined the veracity of observations obtained in cultured fibroblasts aged in vitro to those made in animal tissues in vivo. This paper compares age-related alterations in collagen metabolism measured in cultured cells with previously reported results in the aging rat (Mays et al. (1991) Biochem. J. 276, 307-313). Age-related changes in collagen synthesis in rat skin fibroblasts in vitro over 30 population doublings were determined based on the production of hydroxy-[14C]proline. Degradation of newly synthesized collagen was based on the appearance of free hydroxy-[14C]proline in the culture system. Total protein synthesis rates were based on the incorporation of [14C]proline into proteins. In vitro rates of collagen synthesis decreased 5-fold over 30 population doublings (P < 0.05). Degradation of newly synthesized collagen increased from 33.0 +/- 0.8% (n = 4, SEM) to 45.2 +/- 1.1% (n = 4; P < 0.05) over the same period, with a maximum after 25 population doublings of 55.8 +/- 1.1% (n = 4). Total protein synthesis rates decreased by one-half over 30 population doublings (P < 0.05). The results indicated that collagen production decreased as cells aged in vitro and that this was due to both changes in synthesis and degradation. The results demonstrate that age-related alterations in collagen and total protein metabolism of skin fibroblasts in culture were similar to those reported previously for skin in vivo, suggesting that for studies of these processes, fibroblasts in culture provide an appropriate model.

Changes in collagen metabolism in response to endothelin-1: evidence for fibroblast heterogeneity.

Endothelin-1 (Et-1) is a 21-amino acid peptide primarily synthesized by endothelial cells. It was originally classified as a potent vasoconstrictor but recent evidence suggests that it also possesses a wide variety of non-vascular actions. It stimulates fibroblast and smooth muscle cell proliferation and it has been shown to stimulate fibroblast collagen metabolism. However, studies on its ability to regulate collagen production remain incomplete, and its effect on post-translational processing of procollagen has not been studied. This report details the effect of Et-1 on the rates of procollagen synthesis and degradation in two fibroblast cell lines; human foetal lung (HFL-1) and whole foetal rat fibroblasts (Rat 2). Fibroblast cultures were incubated for 24 hr in the presence or absence of Et-1 before procollagen metabolism was determined by measuring hydroxyproline. Non-collagen metabolism was also determined in these cultures from the uptake of tritiated phenylalanine. Et-1 stimulated procollagen synthesis in HFL-1 fibroblasts and reduced synthesis in Rat 2 cells. The response was dose dependent with the greatest effect at 1.10(-6) M Et-1 for both cell types (155 +/- 6% of control (mean +/- SD, n = 6, P < 0.01) and 61 +/- 4% of control (n = 4, P < 0.01) for HFL-1 and Rat 2 fibroblasts, respectively). Non-collagen protein synthesis was increased to 148 +/- 5% of control (P < 0.05) at 1.10(-6) M Et-1. Non-collagen protein synthesis remained unaffected in the HFL-1 fibroblast cultures. Procollagen degradation, expressed as a proportion of total procollagen synthesis, was decreased in HFL-1 fibroblasts (control, 29 +/- 2%; Et-1, 1.10(-6) M; 21 +/- 2%; P < 0.01), and increased in Rat 2 fibroblasts (control 42 +/- 1%; Et-1, 1.10(-6) M; 49 +/- 1%; P < 0.01). Blocking of the EtA receptor for Et-1, using the receptor antagonist-BQ123, abolished the effect of Et-1 on procollagen metabolism in both cell types. These results suggest that different populations of fibroblasts exhibit heterogeneous responses to Et-1. It is concluded that Et-1 may play an important role in the extent and distribution of fibrosis seen in diseases associated with the overproduction of Et-1.

Age-related changes in rates of protein synthesis and degradation in rat tissues.

It has been hypothesised that a diminished capacity for protein synthesis and degradation underlies a decreased adaptability to environmental stimuli seen during ageing. In this study rates of total protein synthesis and degradation were examined in rats between 1 and 24 months of age. Synthesis rates in heart, lung, skeletal muscle and skin were based on the uptake of [14C]proline into protein when administered with a flooding dose of unlabelled proline. Degradation rates were derived from the difference between protein deposition and synthesis rates. Total protein synthesis rates in 1-month-old animals ranged from 20.4 +/- 1.3% per day (S.E.M.) in skeletal muscle to 39.6 +/- 1.3% per day in lung. In heart, lung and skeletal muscle, synthesis rates decreased 2-fold during the first 6 months of life, while over the same period in skin they decreased 6-fold. Degradation accounted for the bulk of protein synthesised at all ages, and the age-related changes for rates for breakdown closely mirrored those for synthesis. These results, do not support the hypothesis that a general decrease in protein turnover underlies a diminished adaptability in older animals.

Thrombin stimulates smooth muscle cell procollagen synthesis and mRNA levels via a PAR-1 mediated mechanism.

Thrombin is a serine protease involved in haemostasis which exerts a number of cellular effects, including stimulating mesenchymal cell migration, proliferation, and has been implicated both in normal wound healing and pathological conditions associated with hyperproliferation of smooth muscle cells such as atherosclerosis and restenosis. We hypothesize that thrombin, in addition to its proliferative effects, may also influence the deposition of matrix proteins at sites of vascular injury by directly stimulating smooth muscle cell procollagen production. 10 nM thrombin significantly stimulated rat aortic smooth muscle cell procollagen production by 34 +/- 3% compared to media control cells over a 48 h incubation period, and increased steady state alpha1(I) procollagen mRNA levels by up to 104 +/- 22%. These effects are mediated via interaction of thrombin with the PAR-1 receptor since TRAP (Thrombin Receptor Activating Peptide) stimulated procollagen production by 23 +/- 0.5%. In addition, conditioned medium from thrombin-treated cells stimulated procollagen production by 30 +/- 3% suggesting that thrombin is acting via the production and/or release of an autocrine mediator. These data suggest a novel role for thrombin in vascular wound healing and the development of pathological conditions associated with increased connective tissue deposition.

Bleomycin-induced lung injury in the rat: effects of the platelet-activating factor (PAF) receptor antagonist BN 52021 and platelet depletion.

Bleomycin is a highly effective antitumor agent, but pulmonary toxicity, characterized by an acute inflammatory reaction and associated pulmonary edema, limits clinical use of the drug. Platelets and platelet-activating factor (PAF), a membrane-derived phospholipid, have been implicated in the mechanisms that can mediate pulmonary microvascular injury. We sought to investigate the role of PAF in bleomycin-induced lung injury in the rat, using the PAF receptor antagonist BN 52021; and the role of platelets though the use of an anti-platelet antibody. Lung injury was induced by intratracheal bleomycin (1.5 mg) and assessed by measurements of lung wet weight and total pulmonary extravascular albumin space (TPEAS). Bleomycin caused a significant increase in both indices after 48 hr, compared with control animals (p less than 0.05). A single dose of BN 52021 (20 mg/kg orally) significantly reduced the bleomycin-induced increase in lung weight, but not the rise in TPEAS (p greater than 0.05). Increasing the dose of BN 52021 (20 mg/kg/12 hr, orally) had no additional effect. Reducing circulating platelet numbers by approximately 75% had no effect on either the increase in lung weight or TPEAS, observed 48 hr after bleomycin (p greater than 0.05). PAF may partially contribute to the acute inflammatory reaction seen after intratracheal bleomycin in rats.

Long-term changes in mouse lung following inhalation of a fibrosis-inducing dose of 239PuO2: changes in collagen synthesis and degradation rates.

Mice were exposed by nose-only inhalation to 239PuO2, which resulted in an IAD of 1110 +/- 29 Bq. At various times after exposure, rates of collagen metabolism were measured using validated in vivo methods based on the administration of radiolabelled proline, together with a large flooding dose of unlabelled proline and measurement of its incorporation into lung collagen as hydroxyproline. Dramatic increases in both synthesis and degradation rates of collagen were observed. At 54 days after exposure the fractional synthesis rates in experimental mice were almost five times those in controls (control: 3.2 +/- 0.6%/day, 239PuO2-exposed: 14.5 +/- 0.4%/day) and by 300 days synthesis rates, although declining, were still more than double the control values. A similar pattern of change was observed for collagen degradation. The combination of changes in synthesis and degradation rates led to a 60% increase in lung collagen content by 300 days (control: 3.05 +/- 0.24 mg/lung, 239PuO2-exposed: 4.88 +/- 0.42 mg/lung). The data suggest that extensive remodelling of the lung connective tissue matrix occurs during development of fibrosis and that, over long periods of time, small imbalances between synthesis and degradation may result in quite large increases in protein content.

The radiographic detection of acute pulmonary oedema. A comparison of radiographic appearances, densitometry and lung water in dogs.

We have induced acute pulmonary oedema in upright anaesthetized dogs by increasing pulmonary microvascular permeability or by extracellular fluid volume overload in order to determine the sensitivity and specificity of the radiograph to the presence of abnormal lung water. Radiographs were taken before and after development of oedema. At the end of the experiment we removed the inflated lungs and froze them in liquid nitrogen and subsequently examined them macroscopically in the frozen state. The extravascular water/dry lung weight ratio was measured gravimetrically on seven portions of each lung. Finally without the base-line films for comparison. We directly measured the change in opacity of the films with a radiographic densitometer. When a dog's mean extravascular water/g dry lung was increased by more than 35% it was invariably recognized, in one or more zones, as definite oedema. Control dogs were reliably recognized as normal when base-line films were used but the distinction between normal and minor degrees of oedema could not be made without the base-line films. There was a positive correlation between radiological grade and lung water, but a great deal of overlap between grades. Densitometry was not a sensitive or reliable method for diagnosing or quantifying oedema. Oedema was usually associated with a decrease in volume of the lower lung zones.

Pulmonary gene therapy. Realistic hope for the future, or false dawn in the promised land?

Pulmonary gene therapy offers the hope of treatment for conditions such as cystic fibrosis, lung cancer, pulmonary fibrosis and acute respiratory distress syndrome for which current therapy is inadequate. Although initial clinical trials in cystic fibrosis and non-small cell lung cancer have shown promise the results have not been as good as might have been anticipated. However, clinical improvement has been demonstrated in conditions such as haemophilia [82], cardiovascular disease [83], head and neck cancer [84] and X-linked severe combined immunodeficiency disease [85]. The lack of success of pulmonary gene therapy is due, in part on the physical barriers to transfection perfected by the lung to prevent toxicity from inhaled particles, and partly due to the poor transfection efficiency of non-viral systems, and the immunogenicity of viral systems, of gene transfer. The LID vector goes some way to addressing the problems associated with current gene delivery strategies. With continued improvements in the properties of both viral and non-viral gene delivery systems leading to improved transfection efficiency with reduced toxicity, as well as the development of strategies aimed at reducing the physical barriers to pulmonary transfection, and targeting gene delivery systems to the site of injury, it is likely that pulmonary gene therapy will be used successfully to ameliorate a number of devastating pulmonary conditions.

Regulation of matrix turnover: fibroblasts, forces, factors and fibrosis.

Fibroblasts are multifunctional cells that are responsible for matrix homoeostasis, continuously synthesizing and degrading a diverse group of extracellular molecules and their receptors. Rates of turnover of matrix molecules and the proteases that degrade them are normally under the control of diverse chemical and mechanical cues, with cytokines, growth factors, proteases, lipid mediators and mechanical forces playing roles. The maintenance of this homoeostasis is vital to the preservation of normal tissue function and is clearly lost in chronic diseases of the joints, skin and internal organs where destruction and excessive deposition is seen. Current research is focusing on defining the key pathways of activation either in resident fibroblasts, matrix-producing cells derived from circulating fibrocytes, or from transdifferentiation of resident cells. The common downstream signalling pathways are also being defined, as well as the gene interactions leading to altered cell phenotype. The present article reviews these findings and our current concepts of the key molecular events leading to tissue damage and excessive matrix deposition in tissue fibrosis. These studies are leading to an appreciation of the complexity of events with multiple pathways involved, but, as the facts emerge, we are finding promising new ways to treat fibrosis and halt the inexorable progression that is a feature of so many fibrotic and remodelling disorders.

Procollagen type I gene expression and cell proliferation are increased in lipodermatosclerosis.

BACKGROUND: Lipodermatosclerosis (LDS) is characterized by a hardening and hyperpigmentation of lower leg skin as a consequence of chronic venous insufficiency. The degree of skin hardening or fibrosis associated with LDS is proposed to relate directly to skin breakdown and venous ulcer formation as well as to a subsequent delay in ulcer healing. OBJECTIVES: To determine whether elevated procollagen type I gene expression and increased cell proliferation are responsible for the fibrotic changes associated with LDS. METHODS: Skin biopsies were obtained from the legs of patients with varying degrees of chronic venous disease and were assessed for procollagen gene expression by in-situ hybridization and for cell proliferation by immunolocalization of proliferating cell nuclear antigen. RESULTS: The number of cells expressing procollagen type I mRNA (COL1A1) was significantly higher in the dermis of LDS-affected skin compared with samples from the other patient groups. In addition, there was a significant increase in the number of dermal fibroblasts undergoing proliferation in both LDS samples and skin samples prior to LDS changes compared with control samples. However, there was no significant difference in level of inflammation in biopsy samples between patient classes. CONCLUSIONS: These results suggest that enhanced cell proliferation and procollagen gene expression are both involved in LDS development. Furthermore, fibrotic changes may occur in the absence of, or subsequent to, any significant inflammatory response, indicating that additional profibrotic factors produced in the skin as a consequence of chronic venous insufficiency may play a role in LDS formation.

Collagen synthesis and degradation in vivo. Evidence for rapid rates of collagen turnover with extensive degradation of newly synthesized collagen in tissues of the adult rat.

Collagen turnover is now known to occur more rapidly in body tissues than traditionally believed, but the kinetics and mechanisms for degradation are still poorly understood. Here we measure collagen synthesis rates and the proportion of newly synthesized collagen (probably procollagen) which is rapidly degraded, in tissues of the adult rat after injection of [14C]-proline with a large "flooding" dose of unlabelled proline. Incorporation of [14C]-proline into lung, heart, skeletal muscle and skin collagen and its appearance as hydroxy [14C]-proline, free or in small molecular weight moieties, at various times up to one hour, suggested extremely rapid synthesis and degradation for some tissues of the adult rat. Values in heart, lung, skeletal muscle and skin (with the proportion of degradation of newly synthesized collagen shown in parentheses) were 5.2 +/- 0.7%/day (53 +/- 5%), 9.0 +/- 0.7%/day (37 +/- 2%), 2.2 +/- 0.3%/day (38 +/- 7%) and 4.4 +/- 1.3%/day (8.8 +/- 0.5%). These data provide in vivo evidence, which are consistent with the observation in isolated cells, that a proportion of newly synthesized collagen is degraded rapidly, and probably intracellularly, after its synthesis. They also indicate that collagen may be synthesized and degraded rapidly in normal rat tissues, but the mean turnover rates and the proportions of collagen degraded intracellularly vary widely between tissues.

Protein metabolism during bleomycin-induced pulmonary fibrosis in rabbits. In vivo evidence for collagen accumulation because of increased synthesis and decreased degradation of the newly synthesized collagen.

In vivo rates of synthesis and degradation of collagen and noncollagen protein were measured in normal lung and during the first 14 days of bleomycin-induced pulmonary fibrosis in rabbits. Protein synthesis rates were obtained by measuring the incorporation of labeled proline into tissue proteins after the injection of 3H-proline with a large amount of unlabeled proline. Degradation rates were assessed by several independent methods, including measurement of hydroxy-3H-proline in the tissue-free pool as an index of the degradation of newly synthesized collagen. In normal lung, collagen was synthesized and degraded at a rate of 9.5 +/- 1.5%/day compared with a rate of 34.6 +/- 1.9%/day for noncollagen proteins. About one third of the newly synthesized collagen was degraded rapidly after its production. Six days after bleomycin administration the collagen synthesis rate almost doubled and the synthesis rate of noncollagen protein increased by about 35%. At this time the degradation rate of newly synthesized collagen had decreased by about 30%. From this study we conclude: (1) that the lung is an active tissue in terms of protein metabolism with rapid turnover for both collagen and noncollagen proteins, and (2) that an increased rate of synthesis and a decreased rate of degradation contribute to rapid accumulation of collagen in the early stages of pulmonary fibrosis.

Changes in collagen synthesis and degradation during skeletal muscle growth.

The changes in collagen metabolism during skeletal muscle growth were investigated by measuring rates of synthesis and degradation during stretch-induced hypertrophy of the anterior latissimus dorsi muscle of the adult chicken (Gallus domesticus). Synthesis rates were obtained from the uptake of tritiated proline injected intravenously with a flooding dose of unlabeled proline. Degradation of newly synthesized and "mature" collagen was estimated from the amount of hydroxyproline in the free pool as small molecular weight moieties. In normal muscle, the synthesis rate was 1.1 +/- 0.3%/day, with 49 +/- 7% of the newly produced collagen degraded rapidly after synthesis. During hypertrophy there was an increase of about fivefold in the rate of synthesis (P less than 0.01), a 60% decrease in the rate of degradation of newly synthesized collagen (P less than 0.02), and an increase of about fourfold in the amount of degradation of mature collagen (P less than 0.01). These results suggest an important role for degradative as well as synthetic processes in the regulation of collagen mass. They indicate that enhanced degradation of mature collagen is required for muscle growth and suggest a physiological role for the pathway whereby in normal muscle, a large proportion of newly produced collagen is rapidly degraded.

Application of high-pressure liquid chromatography to studies of collagen production by isolated cells in culture.

Techniques for assessing collagen production by cells in culture are usually based on evaluation of uptake of radiolabeled proline into collagen. Although simple in theory, this approach is often flawed because of uncertainties concerning the specific activity of labeled proline in the precursor pool for collagen synthesis. An alternative approach is to assess collagen production directly by measuring hydroxyproline in proteins secreted by cultured cells, although this has been difficult, due to the insensitivity of the methods available. Here we apply high-pressure liquid chromatography using reverse-phase elution of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole derivatives of hydroxyproline to measure collagen production by fibroblasts. The method is easy to perform and allows quantitation of hydroxyproline down to 5 pmol, making it applicable to fibroblasts in 12-well culture plates. Collagen production was shown to be constant over a period of 24 h, with a mean rate of 391 +/- 18 (SE n = 14) ng collagen/10(6) cells/h. Similar values were obtained using thin-layer chromatography and an enzyme-linked immunosorbent assay for type I collagen, but these techniques were judged to be less convenient and required additional assumptions compared with the technique described here in full.

Age-related changes in lung collagen metabolism. A role for degradation in regulating lung collagen production.

Lung collagen levels are determined by a balance between synthesis and degradation, processes known to have rapid rates in young animals. Here, we report age-related changes in lung collagen synthesis and degradation in rats at five ages from 1 month to 2 yr. Synthesis rates were determined after injection of [14C]proline with a flooding dose of unlabeled proline, and its appearance as hydroxy-[14C]proline in protein. To determine degradation of newly synthesized collagen, the appearance of hydroxy-[14C]proline, either free or in low-molecular-weight peptides, was compared with hydroxy-[14C]proline in protein. Fractional collagen synthesis rates decreased from 13.51 +/- 0.54%/day at 1 month to 0.97 +/- 0.14%/day at 2 yr of age (p less than 0.05). Total lung collagen production also fell, but only after 15 months, when it decreased from 2.01 +/- 0.16 mg/day at 15 months to 0.54 +/- 0.10 mg/day at 2 yr of age (p less than 0.05). Fractional rates of total collagen degradation, calculated from the difference between rates of synthesis and rates of collagen deposition, decreased 20-fold from 1 month to 2 yr of age. The proportion of newly synthesized collagen degraded increased from 27.6 +/- 3.2% at 1 month to a maximum of 82.3 +/- 1.1% at 15 months. These results suggest that lung collagen synthesis and degradation occur throughout life, and that degradative pathways may play important roles in regulating collagen production during growth and ageing.